Probing the Structure of Transient Unfolded States with NMR Relaxation Dispersion

描述（由申请人提供）：瞬时天然状态解折叠已被认为是蛋白质错误折叠的初始阶段，随后在许多系统中聚集，并传播成多种疾病。拟议研究的基本目标是使用NMR R2色散技术绘制脱辅基肌红蛋白的天然状态能量景观，应用目标是开发一种新的模型系统，用于理解瞬时蛋白质展开和聚集。R2色散已成功地应用于酶活性中心附近的动力学研究，然而，很少有研究集中在一个轻微扰动的天然状态访问复杂的波动。15 N R2分散体将被应用于理解WT脱辅基肌红蛋白在apoMb保持其天然状态结构的pH值范围内表现出的去折叠转变。激发态的身份将被调查，沿着的pH值和温度依赖性的展开和折叠事件。初步结果表明，在某些条件下，两个网站之间发生交换的原生状态，以及其特征在于熔融球。来自~90个共振的数据将适合于局部两态机制，并评估协同性和机制。全球和集群拟合例程将被应用于提高估计的交换参数，并确定合作结构的单位。多个温度将被用来理解的转变，以及温度依赖性的构象交换的热力学。突变体将用于探测自然状态能量景观中动态波动的F-螺旋的贡献，而非可交换质子的全氘代将用于选择性地探测疏水效应对构象波动的贡献。WT脱辅基肌红蛋白样品然后将分别使用15 N和13 C-甲基R2-分散技术进行调整以产生关于导致无定形（在~ 1 M尿素下）和淀粉样蛋白（在pH 9，>40 ℃下）聚集的过程的动力学信息。将仔细控制样品条件，以保持可溶性样品，同时促进引发聚集的动力学。将进行具体的比较，以揭示在聚集诱导条件之间的展开和再折叠机制的差异，以及那些促进瞬时展开，但没有聚集。为了理解许多聚集机制的初始阶段，有必要了解蛋白质解折叠的过程。由于蛋白质功能通常依赖于瞬时解折叠，因此重要的是要揭示不涉及聚集的解折叠事件与导致形成毒性多聚体物质的解折叠事件之间的关键差异。

DESCRIPTION (provided by applicant): Transient native-state unfolding has been implicated as the initial stage of protein mis-folding and subsequent aggregation in numerous systems that propagate into a multitude of diseases. The basic goal of the proposed research is to map the native-state energy landscape of apo-myoglobin using NMR R2 dispersion techniques, with the applied goal of developing a new model system for understanding transient protein unfolding and aggregation. R2 dispersion has been successfully applied to dynamics studies of enzyme function around the active site, however, few studies have focused on the complex fluctuations accessible to a slightly perturbed native-state. 15N R2 dispersion will be applied to understand the unfolding transitions exhibited by WT apo-myoglobin at a range of pH values for which the apoMb retains its native-state structure. The identity of the excited states will be investigated, along with the pH and temperature dependence of unfolding and refolding events. Initial results suggest that under some conditions a two-site exchange occurs between the native-state, and the well characterized molten globule. Data from ~90 resonances will be fit to a local two-state mechanism, and assessed for cooperativity and mechanism. Global and cluster fitting routines will be applied to improve estimates of the exchange parameters and to identify units of cooperative structure. Multiple temperatures will be used to understand the thermodynamics of the transitions, as well as the temperature dependence of conformational exchange. Mutants will be used to probe the contribution of the dynamically fluctuating F-helix in the native-state energy landscape, while full perdeuteration of non-exchangeable protons will be used to selectively probe the contribution of the hydrophobic effect on the conformational fluctuations. WT apo-myoglobin samples will then be tuned to yield dynamics information about the processes leading to amorphous (at ~1M urea) and amyloid (at pH 9, >40¿ C) aggregation using 15N and 13C-methyl R2-dispersion techniques respectively. The sample conditions will be carefully controlled to maintain soluble samples while promoting the dynamics that initiate aggregation. Specific comparisons will be made to uncover differences in the mechanism of unfolding and refolding between aggregation-inducing conditions, and those which promote transient unfolding but no aggregation. PUBLIC HEALTH RELEVANCE In order to understand the initial stage of many aggregation mechanisms, it is necessary to understand the process of protein unfolding. Since protein function is frequently dependent on transient unfolding, it is important to uncover the key differences between unfolding events that are not implicated in aggregation, with those which result in the formation of toxic multimeric species.